Polyclonal antiserum against the M. oxyfera and NirS enzyme was raised by injecting rabbits with two synthetic peptides: peptide 1 (amino acid position 139–153: PPDKRPTKPEHNRDW) and peptide

2 (amino acid position 520–534: EKARIDDPRIITPTG). Prior to the immunization, an extra amino-terminal cysteine was added to the peptide sequences for the conjugation to the Keyhole limpet haemocyanin (Eurogentec, Belgium). For the M. oxyfera pMMO enzyme, two polyclonal antisera buy Tyrosine Kinase Inhibitor Library targeting α-subunit (pMmoB) were raised. α-pMmoB1 was raised by injection of rabbits with two synthetic peptides: peptide 1 (amino acid position 257–271: QTGRMDTPELKPTTE) and peptide 2 (amino acid position 324–337: DPALFPDSRLKIKVE). Prior to the immunization, an extra amino-terminal SB203580 purchase cysteine was added to the peptide sequences for the conjugation to the Keyhole limpet haemocyanin (Eurogentec). α-pMmoB2 was generated from a heterologously expressed and purified fragment of pmoB in Escherichia coli as described previously (Harhangi et al., 2002), with the following modifications. Two primers were designed on the pmoB sequence; a forward primer on nucleotide position 790 (CCCGAACTGAAGCCCACGACAGAG) and a reverse primer on nucleotide position 1188 (GCCGCCGACCTCAACAATTTGTCTG). A stop codon

(TAA) was included in the reverse primer so as to express only an N-terminal His-tag. For directional cloning, restriction sites EcoRI and NotI were included in the forward primer and XhoI in the reverse primer. An additional nucleotide (T) was added between EcoRI and NotI so as to bring the sequence in frame. pET-30a(+) (Novagen, Germany) was used as the expression vector. Rosetta cells (Novagen) were used as the expression host. The heterologously expressed protein fragment (amino acid position 264–396) was purified using the HIS-Select® HF nickel affinity gel column (Sigma, The Netherlands) under denaturing conditions using the protocol dipyridamole provided by the manufacturer. The identity of the expressed protein fragment was verified by MALDI-TOF MS peptide mass fingerprinting of a tryptic digest of the purified

protein fragment (Harhangi et al., 2002). For each antiserum, two rabbits were immunized using a 3-month immunization protocol. The antisera from both rabbits were pooled and affinity-purified (Eurogentec). The affinity-purified antisera (α-NirS, α-pMmoB1 and α-pMmoB2) were used as the primary antisera in immunoblot analysis and immunogold localization as described later. Approximately 2 g of cells (wet weight) was taken from the M. oxyfera enrichment culture. The cells were washed three times with 20 mM phosphate buffer pH 8.0 and resuspended in a medium containing 20 mM sodium phosphate and 50 mM sodium pyrophosphate pH 8.0. Cells were broken by sonication. Cell debris was removed by centrifugation (6000 g, 15 min, 4 °C), and the supernatant was collected as whole-cell extract.